Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway

Abstract

Background: The automation of modular cloning methodologies permits the assembly of many genetic designs. Utilising characterised biological parts aids in the design and redesign of genetic pathways. The characterisation information held on datasheets can be used to determine whether a biological part meets the design requirements. To manage the design of genetic pathways, researchers have turned to modelling-based computer aided design software tools.

Result: An automated workflow has been developed for the design and build of heterologous metabolic pathways. In addition, to demonstrate the powers of electronic datasheets we have developed software which can transfer part information from a datasheet to the Design of Experiment software JMP. To this end we were able to use Design of Experiment software to rationally design and test randomised samples from the design space of a lycopene pathway in E. coli. This pathway was optimised by individually modulating the promoter strength, RBS strength, and gene order targets.

Conclusion: The use of standardised and characterised biological parts will empower a design-oriented synthetic biology for the forward engineering of heterologous expression systems. A Design of Experiment approach streamlines the design-build-test cycle to achieve optimised solutions in biodesign. Developed automated workflows provide effective transfer of information between characterised information (in the form of datasheets) and DoE software.

Keywords: Automation workflow; Datasheets; Design of Experiment (DoE); Synthetic biology.

Fig. 1

Diagram of the automated workflow for the designing and building of heterologous metabolic pathways; To aid in the development of new novel metabolic pathways in microbial hosts a design-build framework was established. To build a metabolic pathway, information from data repositories is fed into BioCAD software. BioCAD constructs a comprehensive virtual assembly of every potential pathway design. Once designed, metabolic pathways are constructed and tested using automated liquid handling robotics. b To achieve an automated design-build framework with minimal human involvement, in-house software tools and data models have been developed. These tools have been installed into automated pipeline, represented by the red or yellow arrowed lines. The grey and coloured arrowed lines represent the flow of information through the process development of a heterologous metabolic pathway. a A workflow and data model has been developed to capture and disseminate part information to an online datasheet repository known as SynBIS. c An in-house software tool has been developed to convert SBOL serialised datasheets to readable CSV files to input biological part information into the JMP DoE software tool. Screenshots of the software and CSV file can be seen in the supplementary information (Additional file 1: Figure S1). d JMP DoE software tool can be utilised to design biosynthetic genetic pathways. The software tool AMOS has been developed to coordinate the building of selected samples from the design space created by JMP in liquid handling robots

Fig. 2

The biological parts utilised for combinatorial construction of a lycopene biosynthetic pathway. a The carotenoid lycopene is produced in E. coli through a heterologous biosynthetic pathway composed of three enzymes (CrtE. CrtB and CrtI). The heterologous proteins use FPP, which is a product of MEP pathway, as a precursor. b A complete combinatorial library totalling 810 pathway configurations can be designed by varying the promoter and RBS parts and by varying the order of pathway genes and RBS parts. Through the application of statistical modelling by DoE software, the designed library was reduced to 88 representative constructs, A table displaying part information of each construct is displayed in the supplementary information ( Additional file 1: Table S1). The pathway library was assembled and expressed in E. coli DH5α to test lycopene titres. The relative effects of the different design factors had on the titres level of lycopene were modelled using JMP. The model identified biological parts which effected titre levels, this can help aid future designing of the lycopene biosynthetic pathway.

Fig. 3

Lycopene production by different E. coli designs. 88 assemblies of the lycopene biosynthesis pathway from the design space were tested in E. coli. Of the 88 constructs, 46 produced colonies after transformation, of which 41 produced detectable lycopene content. Each clone was grown for 24 h in LB broth before lycopene content was determined as mg lycopene per dry cell weight (DCW). Error bars represent the standard deviation across three independent biological replicates